Abstract Viruses have historically revealed novel cellular processes, including mRNA nuclear export pathways. Here we study a recently identified pathway usurped by influenza virus mRNAs to mediate their nuclear export. This process is driven by the viral NS1 protein interaction with cellular and other viral proteins. NS1 is a major virulence factor that inhibits cellular antiviral response and promotes viral gene expression. In this proposal, molecular mechanisms of NS1 functions that mediate viral mRNA nuclear export and the mechanism of action of a novel small molecule inhibitor of viral mRNA nuclear export will be examined. In particular, our published and preliminary structure-function data show that NS1 inhibits the export of cellular (host) mRNA from the nucleus to the cytoplasm by interacting directly with the heterodimeric host mRNA export receptor NXF1-NXT1. Moreover, NS1 prevents NXF1-NXT1 docking to the nuclear pore complex. However, NS1 is also critical for nuclear export of at least some of the essential influenza mRNAs, even though these also require NXF1. In Aim 1 of the current proposal, this paradoxical observation will be investigated by determination of the molecular mechanisms that mediate influenza mRNA nuclear export. A combination of genetics, single-molecule RNA- FISH (smFISH), in vitro and cell-based binding assays, and a viral mini-genome system will be used to explore how NS1 promotes the export of viral mRNAs. Aim 2 will address target identification/validation of a novel inhibitor of viral mRNA nuclear export. It is based on the identification of a cellular NS1-binding protein that mediates nuclear export of viral mRNAs and of a subset of cellular mRNAs, but not bulk cellular mRNA. This provided an avenue to target influenza virus without compromising bulk cellular gene expression. We designed and performed a high-content, image-based chemical screen and identified a novel inhibitor of viral mRNA export that exhibited no significant activity towards bulk cellular mRNA. This small molecule inhibits virus replication at non-toxic concentrations. Preliminary data indicate that this small molecule targets the cellular TAOK2 kinase. The focus of Aim 2 will be on target validation and identification of potential additional targets of this novel inhibitor of mRNA export using photo-crosslinking based chemical approaches, and functional genetic, biochemical, and imaging approaches. By determining how NS1 influences mRNA export and how this process can be regulated by a small molecule, we will uncover fundamental new mechanisms and regulation of a noncanonical mRNA export pathway. Additionally, this compound may serve as a lead for developing antivirals.